The present invention relates to gene constructs suitable for expressing agents to protect a plant against pathogens and the suitable proteins for such plant protection. These agents are named xe2x80x9cmolecular pathogenicidesxe2x80x9d. This invention is related to the genetic engineering of plants and to means and methods for conferring pathogen resistance on a plant using a gene or genes encoding: a pathogen specific antibody and a pathogen specific antibody including a toxic activity which blocks stages of the pathogen life cycle, pathogen replication or pathogen movement within a plant or pathogen transmission from plant to plant. The means and methods are given for soluble expression of recombinant antibodies, antibody fusion proteins and antibody protein complexes in different plant cell compartments or the immobilisation of recombinant antibodies, antibody fusion proteins and antibody protein complexes in cellular membranes in different orientations and the display of recombinant proteins on the plant cell membrane. This invention also describes novel methods and protein binding partners for assembling protein complexes from individual polypeptide chains during expression of these proteins in vivo. Also given are the methods for activation of the molecular pathogenicides by in vivo proteolytic cleavage.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturers specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.
Plant disease constitutes a major and ongoing threat to human food stocks and animal feed. Most crop plants are regularly exposed to one or more pathogen(s) that can cause incredible damage resulting in substantial economical losses every year. Attack by pathogens, such as viruses, bacteria, fungi, nematodes and insects and is a severe economic problem, which impacts all economically important crops, for example rice, soybean, sweet potato, wheat, potato, grape, maize and ornamental plants. Current protective measures rely heavily on chemical control measures for pathogen vectors, which have undesirable environmental consequences.
A more effective approach to protecting plants from pathogen attack is to create plants that are endogenously resistant to pathogens. However, plant breeders have limited sources of resistance genes against plant diseases. This can now be achieved using genetic engineering techniques, by providing the plant with genetic information required for affecting the pathogens and for being resistant to the disease caused by the pathogen. For example, in the case of a viral pathogen, the host plant is resistant if it has the ability to inhibit or retard the replication of a virus, the symptoms of viral infection or the life cycle of the virus, including its transmission. xe2x80x9cResistantxe2x80x9d is the opposite of xe2x80x9csusceptiblexe2x80x9d and may be divided into three levels:
1) Full,
2) Medium,
3) Partial resistance,
A plant may be considered fully resistant when it shows no symptoms on infection and there is no evidence of pathogen replication or reproduction. The host plant may be resistant to the establishment of infection, pathogen reproduction and/or pathogen movement and transmission.
In recent years, the advances in plant molecular virology have enhanced the understanding of pathogen genome organisation and gene function. Moreover, genetic engineering of plants for virus resistance has recently provided new strategies for control of viral disease (Baulcombe, 1994), (Gadani et al., 1990), (Wilson, 1993). The following genes were expressed in transgenic plants in order to confer resistance: viral coat proteins, non-structural proteins of viral genomes, viral anti-sense transcripts, viral satellite RNAs, ribozymes and interferon genes (Baulcombe, 1994), (Gadani et al., 1990), (Wilson, 1993), (Harrison et al., 1987), (Namba et al., 1991), (Anderson et al., 1992). Although most of these approaches have been effective for attenuating infections, resistance was not complete and confined to a small spectrum of viral pathogens (Falk and Bruening, 1994), (Wilson, 1993) and bears significant risks (Palukaitis and Roossinck, 1996).
The major disadvantages of these methods are:
1) Host range is limited.
2) Pathogen range is limited.
3) Resistance is partial and though symptoms are delayed infection still results in the disease.
4) Resistance could be broken in case of coat protein mediated resistance and ribozyme mediated resistance.
5) Expression of viral proteins can lead to enhanced pathogen activity. For example, in the case of viral coat protein mediated resistance, cross encapsidation of mild non-pathogenic strains of virus by the expressed coat protein can occur which then leads to development of a more severe disease.
An alternative way to protect plants against pathogen infection is the generation and expression of recombinant antibodies (rAbs), which are often referred to as xe2x80x9cPlantibodiesxe2x80x9d. Pathogen-specific recombinant antibodies targeted to different compartments of plant cells or different plant organs overcome many of the problems mentioned before and confer a broader spectrum of resistance to disease (Baulcombe, 1994). To achieve this, recombinant antibodies (Plxc3xcckthun, 1991), (Winter and Milstein, 1991) against the target proteins have to be generated by cloning the corresponding antibody heavy and light chain genes from hybridoma cells, synthetic, semi-synthetic and immunocompetent phage display or ribosome display libraries; or by the generation of fully synthetic designer antibodies. This is followed by subsequent modification and rAb expression in different compartments of heterologous hosts such as bacteria, yeast, algae, baculovirus infected insect cells, mammalian cells and plants. For example, antibodies and antibody-fusion proteins binding to conserved functional domains of viral coat proteins, movement proteins, replicases or transmission factors can be used to inactivate such targets inside or outside the plant cell through immunomodulation. The feasibility of expressing recombinant antibodies (Plxc3xcckthun, 1991), (Winter and Milstein, 1991) for the generation of resistance has been shown recently for both animal (Chen et al., 1994), (Duan et al., 1994), (Marasco et al., 1993) and plant viruses (Tavladoraki et al., 1993), (Voss et al., 1995), (Zimmermann et al., 1998). Single chain antibody fragments derived from monoclonal antibodies (scFvs) (Bird et al., 1988) directed against Rev (Duan et al., 1994) and gp120 (Chen et al., 1994) (Marasco et al., 1993) of HIV, inhibited HIV-replication, virion assembly and syncytia formation when expressed intracellularly, or within the ER of human cells.
Interestingly, intracellular expression of an scFv specific for the artichoke mottled crinkle virus coat protein in transgenic Tobacco caused a reduction of infection and a delay in symptom development (Tavladoraki et al., 1993). Targeting of TMV-specific full-size antibodies to the intercellular space of Tobacco plants inhibited viral infections up to 70% (Voss et al., 1995). In the latter case, plant produced antibodies showed the same specificity and affinity for TMV (Fischer et al., 1998) as the parental murine antibody. Cytosolic expression of an engineered scFv derived from this anti-TMV antibody yielded fully resistant Tobacco plants, even under systemic infection conditions (Zimmermann et al., 1998). These studies demonstrate the potential of heterologously expressed recombinant antibodies to combat pathogens via intra- or extra-cellular modulation of pathogen proteins.
Plant cells can synthesise large amounts of antibodies that are functionally indistinguishable from the source monoclonal. For example, full-size antibodies (During et al., 1990), (Hiatt et al., 1989), (Voss et al., 1995), Fab-fragments (De Neve et al., 1993), scFvs (Owen et al., 1992; Zimmermann et al., 1998), (Tavladoraki et al., 1993), scFv fusion proteins (Spiegel et al., Plant Science 149 (1999), 63-71), bispecific scFv (Fischer et al., 1999) and dAbs (Benvenuto et al., 1991) have been successfully expressed in Tobacco, Potato (Schouten et al., 1997) or Arabidopsis, reaching expression levels as high as 6.8% of the total protein (Fiedler et al., 1997). Targeting of recombinant antibodies by exploiting known protein trafficking signal sequences now permits rAb expression in the cytoplasm (scFv fragments (Tavladoraki et al., 1993; Zimmermann et al., 1998)), the endoplasmic reticulum (Fiedler et al., 1997), chloroplasts (During et al., 1990) and the intercellular space (Benvenuto et al., 1991; De Neve et al., 1993; Voss et al., 1995; Zimmermann et al., 1998) (full-size, Fab fragments, scFvs and single domain Abs). These results demonstrate the flexibility of the plant system to express any recombinant antibody or recombinant antibody fragments in almost all plant compartments, using targeting sequences that also may be from plants or derived from other eukaryotes.
The advantage of targeted protein expression is that the rAbs can be expressed where the pathogen is most vulnerable and where they will have the maximal protective effect. In patent application WO 96/09398 the use of antibody-fusion proteins as agents for controlling crop disease caused by pathogens is proposed.
The antibody delivers a toxin which kills the pathogen in transgenic plants or when expressed or applied as an external immunotoxin. WO 96/09398 is focussed on recombinant Ab-fusion proteinsxe2x80x94single polypeptides that are either genetically, chemically or xe2x80x9cbiochemicallyxe2x80x9d linked to form an immunotoxin. However, WO 96/09398 does not provide proof of principle for antibody mediated pathogen resistance and it was doubtful whether any of the hypothetical examples in WO 96/09398 would work to the extent that a protection of plants against pathogen attack can be obtained sufficient to comply with the needs of the breeders and farmers. Thus, there is still a need of means and methods for conferring antipathogenic/predator characteristics to transgenic plants.
The objective of this current patent application is to provide means and methods for protecting plants, in particular monocotyledonous and dicotyledonous agricultural crops and ornamental plants, against pathogens in a more effective and environmentally sensitive manner. Accordingly, the solution to the technical problem is achieved by providing the embodiments characterised in the claims.
As will be described hereinbelow, the above-mentioned objective is met according to the invention by any one of the following or any combination of the following inventions: i) the expression of pathogen specific recombinant antibodies and parts thereof, or ii) by fusing antibodies or parts thereof to toxins, proteins, or enzymes having activity against the pathogens or to the effective parts of these toxins or enzymes, and then expressing these fusion proteins, or iii) by assembling protein complexes composed of an antibody or fragment thereof in vivo using the novel binding proteins described here and or iv) including a specific protease sensitive sequence, that is cleaved (e.g. in the presence of the pathogen or in a specific plant cell compartment) to release and or activate the toxic activity of any of the recombinant proteins in i) to iii), and or v) targeting or integrating any of the recombinant proteins in i) to iv) to cell membranes in any orientation. These agents are also named xe2x80x9cmolecular pathogenicidesxe2x80x9d. Thus, in one aspect the present invention relates to a fusion protein comprising
(a) at least one binding domain specifically recognising an epitope of a plant pathogen; and
(b) at least one further domain comprising a protein or peptide sequence which is toxic to the pathogen or detrimental to its replication, transmission or life cycle.
Said domains can be linked by covalent or non-covalent bonds. In a preferred embodiment of the fusion protein of the invention said binding domain comprises an antibody, a T-cell receptor, a pathogen specific receptor, a peptide specific for an epitope of a pathogen, or at least the binding site of any one of those. In another aspect, the invention relates to membrane associated binding domains and further domains, respectively, as defined herein.
The fusion proteins composed of a pathogen specific antibody and toxin molecule can be made by fusing the respective parts by genetic or biochemical means. In addition, the chimeric protein can preferably be assembled in vivo from its parts by the plant or via expression in the organisms"" endogenous protein machinery. In a particularly preferred and advantageous embodiment of the invention, these domains or parts thereof, fusion proteins or protein complexes can also be targeted to organelles and plant cell compartments or immobilised and membrane anchored by the addition of signal sequences and or membrane anchors. The recombinant molecular pathogenicide protein preferably contains specific protease cleavage sequences that are cleaved in vivo, by a plant and/or a pathogen specific protease(s), to release and or activate the toxic agent(s), or parts thereof, upon infection.
The fusion protein of the present invention can further comprise a carrier protein suitable for delivering the fusion protein or its domains into a host cell, preferably plant cell or a cellular compartment thereof. Furthermore, the fusion protein of the present invention can comprise a fluorophore such as green fluorescent protein fused to at least one of the above-described domains the fusion protein consists of. In a further aspect, the present invention relates to a pathogenicide comprising at least one binding and/or further domain as defined herein and a cellular targeting sequence and/or membrane localisation sequence and/or motif that leads to membrane anchoring. Preferably, the membrane localisation sequence is proteolytically sensitive.
Suitable membrane anchor sequences, enabling the integration of secretory recombinant antibody fusion proteins and parts thereof in the plasma membrane, include the human T cell receptor transmembrane domains (Gross and Eshhar, 1992), glyco-phosphatidyl inositol (GPI) anchors (Gerber et al., 1992), immunoglobulin superfamily membrane anchors, tetraspan family members (Tedder and Engel, 1994; Wright and Tomlinson, 1994) and any transmembrane sequence(s) from a known protein or synthesised sequences that have a similar function and can be included in the target protein by recombinant DNA technology. Fusion of a protein to these sequences would permit display of the recombinant protein on the lumenal face of organelles of the secretory or endocytic pathway or the plant cell membrane. This has the advantage that the recombinant protein can be targeted to the intracellular space where many pathogens are most vulnerable.
In addition, the antibodies or parts thereof, or the recombinant antibody fusion proteins, or parts thereof, may be targeted to cell membranes where they could face the cytosolic side of the membrane. Suitable targeting sequences for cytoplasmic display, include the transmembrane domains of: KAR1, for nuclear membrane integration (Rose and Fink, 1987), middle-T antigen (Kim et al., 1997), for plasma membrane integration and cytochrome b5, for ER membrane integration (Kim et al., 1997). C-terminal linkages to fatty acids using consensus amino acid sequences leading to post translational prenylation, farnesylation, palmitoylation, myristoylation or ankyrin sequence motifs can also be used. This cytoplasmic display method has the significant advantage that the recombinant proteins can be localised at the site of intracellular pathogen replication, where they will have the most potent effect. In addition, membrane localisation of proteins stabilises the protein and reduces the effect of C-terminal protein degradation in vivo. Preferably, the pathogenicide of the invention comprises the fusion protein described herein.
In a particularly preferred embodiment, the present invention relates to the described pathogenicides wherein said binding domain(s) and/or said further domain(s) are capable of self assembly in vivo.
In a further embodiment, the present invention relates to a polynucleotide encoding a fusion protein or pathogenicide of the invention. Thus, the invention relates to one or more gene constructs that encode a nucleotide sequence encoding an antibody or part thereof which is specific for a pathogen and in the case of fusion proteins, for a nucleotide sequence encoding a protein, enzyme or peptide which has detrimental effects on a pathogen and ideally is toxic to the pathogen. This invention includes antibodies specific for the pathogen and/or for host proteins utilised by the pathogen during its life cycle. This invention also relates to chimeric proteins that consist of an antibody, antibodies or parts thereof, which are specific for a pathogen, and a protein or peptide which has detrimental or ideally toxic effects on the pathogen and which has been constructed by biochemically linking the antibody or parts thereof to the toxin. Furthermore, the present invention relates to a vector comprising the polynucleotide of the invention. Said vector can comprise separate polynucleotides encoding at least one of said binding domain(s) and/or said further domain(s) of the above-described fusion protein. In addition, the present invention relates to a composition comprising vectors wherein each vector contains at least one polynucleotide encoding at least one binding domain and/or at least one further domain of the fusion protein or the pathogenicide of the invention; and wherein the expression of at least two of said polynucleotides results in the production of said fusion protein or said pathogenicide or assembly of the same in vivo.
In a preferred embodiment of the vector or the composition of the invention the polynucleotide is operatively linked to regulatory sequences allowing the expression of the fusion protein, pathogenicide or the domains thereof in a host cell. Said regulatory sequence can be a constitutive, chimeric, tissue specific or inducible promoter.
Furthermore, the present invention relates to a host cell comprising any one of the above-described polynucleotides, vectors or vectors of the compositions.
In another embodiment the present invention relates to a method for the production of a molecular pathogenicide comprising:
(a) culturing the host cell of the invention under conditions suitable for the expression of the polynucleotide; and
(b) recovering the fusion protein, pathogenicide or the domains thereof from the culture.
The present invention also relates to a molecular pathogenicide obtainable by the method of the invention or encodable by the polynucleotide of the invention.
This invention also relates to in vivo assembled protein complexes composed of one or more discrete polypeptide chains, encoded by separate nucleotide sequences on one or more constructs, that are assembled by the plant or expression organisms protein synthesis machinery into a protein complex.
Furthermore, the present invention relates to a method for the production of pathogen resistant transgenic plants, plant cells or plant tissue comprising the introduction of a polynucleotide or vector of the invention or the vectors of the composition of the invention into the genome of a plant, plant cell or plant tissue.
The present invention also relates to a transgenic plant cell which contains stably integrated into the genome a polynucleotide or vector of the invention or the vectors of the composition of the invention or obtainable according to the method of the invention.
In addition, the present invention relates to a transgenic plant or plant tissue comprising the above-described plant cells or obtainable by the method of the invention. Encompassed are also the transgenic plants wherein the fusion protein or pathogenicide are made functional against pathogens by in vivo assembly after co-transformation of at least two independent plant expression constructs or after sexual crossing to form hybrid offspring from two parental plants expressing one or more of the domains of the fusion protein or the pathogenicide, or any other form of genetic recombination. Preferably, the transgenic plant of the invention displays improved resistance against a pathogen that the wild type plant was susceptible to.
Furthermore, the present invention relates to harvestable parts and propagation material of a plant of the invention comprising plant cells of the invention.
In a still further embodiment, the present invention relates to a kit comprising any one of the described fusion proteins, pathogenicides, polynucleotides, compositions or molecular pathogenicides of the invention.
In another embodiment the present invention relates to the use of the described antibodies, fusion proteins, polynucleotides, vectors, compositions and molecular pathogenicides of the invention in agriculture for the protection of a plant against the action of a pathogen.
Some aspects of the present invention will be described herein below in more detail.
The term xe2x80x9cbinding domainxe2x80x9d is used to denote polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given epitope or epitopes. Preferably, said binding domain is an antibody or a binding site thereof. The antibodies may be generated by hybridoma technology, or ribosome display, or phage display, of natural naxc3xafve origin, or immunised origin, semi-synthetic or fully synthetic libraries. The term xe2x80x9cantibodyxe2x80x9d is also used to denote designer antibodies. These antibody, polypeptides are encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind the given epitope or epitopes. The recognised immunoglobulin genes include the kappa and lambda light chain genes, the mu, delta, gamma, alpha and epsilon constant regions as well as all immunoglobulin variable regions from vertebrate, camelid, avian and pisces species. The term antibody, as used herein, includes in particular those antibodies synthesised or constructed de novo using recombinant DNA methodology, such as recombinant full-size antibodies, dimeric secretory IgA antibodies, multimeric IgM antibodies, F(abxe2x80x2)2-fragments, Fab-fragments, Fv-fragments, single chain Fv-fragments (scFvs), bispecific scFvs, diabodies, single domain antibodies (dAb), minibodies and molecular recognition units (MRUs). Antibody sequences may be derived from any vertebrate, camelid, avian or pisces species using recombinant DNA technology, or also by using synthetic, semi-synthetic and naive or immunocompetent phage and ribosome display libraries, gene shuffling libraries, and fully synthetic designer antibodies. In this invention, the antibodies are generated against specific pathogen or host plant epitopes that are involved in the pathogen replication, reproduction or life cycle.
The term xe2x80x9cpathogenxe2x80x9d is used to denote viral or virus like organisms, bacteria, mycoplasmas, fungi, insects or nematodes that affect the germination of seed, growth, development, reproduction, harvest, yield or utility of a plant. The term xe2x80x9ctoxicxe2x80x9d refers to an activity, which may be peptide or polypeptide encoded, that affects the reproduction or replication of a pathogen and/or any stages of its life cycle. In the case of viral pathogens, this includes entry into the plant, viral uncoating and disassembly, viral replication, viral assembly, cell to cell and long distance movement and the development, spread, or life cycle of the virus. Suitable toxic activities include RNAse (Leland et al., 1998) and DNAse, ribosome inactivating proteins (Barbieri et al., 1993), (Girbes et al., 1996), (Hartley et al., 1996) and or toxins with antimicrobial activity (Dempsey et al., 1998). Antibodies or recombinant proteins in themselves are also considered toxic when they affect the pathogen by binding to pathogen and or host proteins that are utilised by a pathogen during its replication, reproduction, life cycle or transmission. For example, a fusion protein composed of a virus specific antibody and a viral coat protein will interfere with virus reproduction by both binding to the virus and by disrupting viral assembly or disassembly in the host cell.
The term xe2x80x9cmolecular pathogenicidexe2x80x9d refers to the antibodies and proteins described in this application, which have toxic effects on pathogen(s) either as single fusion proteins, when expressed in combination with other proteins, or when expressed as part of protein complexes that are assembled in vivo.
Monoclonal antibodies (Kxc3x6hler and Milstein, 1975) can be raised against almost any epitope or molecular structure of a pathogen or host protein using several techniques. The most common method is the hybridoma technique starting with immunocompetent B lymphocytes from the spleen or thymus which are obtained after immunisation with native antigen, recombinant antigen, antigen fusion proteins, antigen domains or by in vitro or genetic immunisation. In addition, recent advances in molecular biology techniques now permit the use of cloned recombinant antibody fragments and antibodies derived from mice and other organisms than the mouse. Suitable recombinant antibody fragment(s) include the complete recombinant full-size antibodies, dimeric secretory IgA antibodies, multimeric IgM antibodies, the F(abxe2x80x2)2 fragment, the Fab-fragment, the Fv-fragment, single chain antibody fragments (scFvs), single binding domains (dAbs), a bivalent scFv (diabody) (Poljak, 1994), minibody (Carter and Merchant, 1997), bispecific scFv antibodies (Plxc3xcckthun and Pack, 1997) where the antibody molecule recognises two different epitopes, (which may be from the pathogen or the host or both the pathogen and the host), triabodies and any other part of the antibody such as, molecular recognition units (MRUs), which show binding to the target epitopes. Genes encoding these suitable recombinant antibody fragment(s) may be derived from vertebrates, camelids, avian or pisces species.
Also, single chain antibodies that have affinities for pathogen or host structures and proteins can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries, which can be constructed from synthetic, semi-synthetic or naxc3xafve and immunocompetent sources (Plxc3xcckthun, 1991; Winter et al., 1994; Winter and Milstein, 1991). Phage display and suitable techniques can be used to specifically identify antibodies, or fragments thereof, with the desired binding properties. Using recombinant antibody technology it is possible to identify antibodies or fragments that are highly specific for a single pathogen, or which recognise a consensus epitope conserved between several pathogens, where the antibodies will have a broad specificity against pathogens. The durability and effect of antibody mediated resistance can be improved by i) recombinant antibody affinity maturation, ii) CDR randomisation and selection, iii) stabilisation by framework optimisation of a selected pathogen specific antibody, iv) bi-specific antibody expression, v) the generation of antibody fusion proteins, or vi) the expression of antibodies in combinations with others that may potentiate their individual effects. For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage displayed antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of a pathogen (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The recombinant antibodies can be identified and utilised according to methods that are familiar to anyone of ordinary skill in the art.
This invention describes antibodies or fragments thereof which recognise structures of the pathogen or host plant and directly or indirectly lead to resistance or partial resistance when expressed alone or when expressed as chimeric fusion protein coupled to a toxic activity or when expressed and assembled in viva with a toxic activity to form an in vivo assembled molecular pathogenicide protein complex. Antibodies can be generated that recognise pathogen-specific epitopes or host plant-specific epitopes which have a role in the life cycle of a pathogen. Suitable antibodies for engineering viral resistance include, but are not limited to, those binding to conserved functional domains of viral coat proteins, movement proteins, or replicases and are an approach to obtain broad-spectrum resistance and reduce the environmental risks by inactivating the targets inside and/or outside the plant cell through immunomodulation. The feasibility of this approach has been recently shown for both animal (Chen et al., 1994), (Duan et al., 1994), (Marasco et al., 1993) and plant viral resistance (Tavladoraki et al., 1993), (Voss et al., 1995), (Zimmermann et al., 1998). These antibodies or fragments thereof may be inactivating in themselves or in combination with one or more other antibodies, or a toxin, or in combination with a carrier, transmembrane domain or signal peptide. Importantly, plant pathogen resistance can be enhanced by the co-expression of multiple antibodies.
In a particular preferred embodiment, the present invention relates to one of the above-described antibodies wherein the antibody or a derivative thereof is capable of binding to the functional domain of a viral movement and/or replicase protein. As could be surprisingly demonstrated in Examples 5 and 6, antibodies directed against a viral movement and replicase, respectively, can be used to engineer enhanced resistance against the virus the movement and replicase gene are derived from. The advantage of using the movement or replicase protein as a target for the antibody or a functional equivalent binding protein is that the functional domains within the movement protein and the replicase can be expected to be highly conserved among different viruses. Thus, the expression of an antibody directed against such a conserved epitope of, for example, the movement protein of TMV can also be expected to be effective against related viruses. Furthermore, due to the conservation of the functional domains in these two viral proteins, a further advantage is that the heterogeneity within one single virus group should not be as high as for, e.g., the coat protein. Thus, the finding of the present invention that the movement and replicase protein of a virus are accessible to antibody targeting within a plant cell, a novel concept for the generation of virus resistant plants became feasible. It is therefore, that in one separate aspect the present invention relates to such antibodies for engineering virus resistance in plants. Viruses that can be the target of this approach are any that use movement proteins during infection as well as all viruses that encode a replicase gene. This can be expected to be effective because viral movement is a common feature of many viral infections (McLean et al., Trends Microbiol. 1, (1993), 105-9) and replicases are essential for viral pathogenesis. The importance of approaches targeting these proteins is underscored by the fact that expressing wild type or defective versions of movement or replicase proteins often results in resistance (Beachy, (1997), Curr. Opin. Biotechnol. 8:215-220). Transgenic plants expressing defective mutant TMV movement protein are resistant to multiple viruses, presumably because of disruptions in intercellular viral movement (Cooper et al., (1995), Virology 206, 307-313) and replicase expression is an effective resistance strategy (Anderson et al., (1992), Proc. Natl. Acad. Sci. USA 89:8759-8763; Baulcombe, (1994), Trends Microbiol. 2:60-63; Brederode et al., (1995), Virology 207:467-474; Nguyen et al., (1996), Proc. Natl. Acad. Sci. USA 93:12643-12467; Rubino and Russo, (1995), Virology 212:240-243).
A disadvantage of the current antibody mediated resistance approaches may be the choice of viral coat proteins as target. Plant viral coat proteins have a broad structural diversity and this can restrict the effect of the expressed antibodies to a small range of viruses and under selective stress, the viral coat protein sequence can alter without loss of function. Generation of recombinant antibodies directed against conserved functional domains of viral replicases and movement proteins may provide a better route for obtaining pathogen resistant plants with a broad-spectrum resistance against viruses. The antigen for producing any one of the above-described antibodies can be derived from naturally occurring movement or replicase proteins or fragments thereof or can be recombinantly produced, chemically synthesized and/or derivatized by methods well known to the person skilled in the art some of which are also further discussed herein. In view of the above, the invention also relates to polynucleotides encoding the above-described antibodies, vectors comprising the same and host cells transformed therewith. Suitable vectors, host cells and strategies for the expression of recombinant antibodies in plants are described herein and can be easily adapted from any one of the other embodiments described herein.
Toxins include all proteins and peptides that have a detrimental or toxic effect on a pathogen during its life cycle and/or an effect on the pathogen during plant infection or pathogen replication, spread or transmission. This includes toxins that specifically kill an infected host cell and so limit the spread and development of a disease.
Suitable toxins include the following:
toxic peptide(s) which are specific for the pathogen and mediates toxicity e.g. by membrane permeabilisation based on alteration of membrane potential (Ham et al., 1994; Sangster, 1997).
blocking peptides which bind to structural or non structural pathogen proteins, or nucleic acid motifs, and inhibit pathogen function, growth, development or toxicity to the host (Hayakawa, 1991; Silburn et al., 1998).
peptide mimics that bind to pathogen or host protein motifs and that modulate or block the pathogen""s replication, e.g. peptide derivatives of proteinase inhibitors that play a physiological role as inhibitors of viral replication and can be used as antiviral agents (Bjorck et al., 1990), (Bjorck et al., 1989).
binding domains, such as antibodies defined above specifically recognising an epitope of a plant pathogen.
peptide mimics that bind to pathogen or host protein motifs and that modulate or block the pathogen""s movement within the host plant. As an example, the BC peptide, which mimics the nuclear localisation signal region of HIV-1, reduces HIV-1 production by 75% when expressed in infected dividing cultured human T-cells (Friedler et al., 1998).
toxins which kill the host cell where the pathogen is replicating and has penetrated the cytosol (Barbieri et al., 1993; Hartley et al., 1996; Madshus and Stenmark, 1992), for example (Ribosome inactivating proteins) RIPs which enter the cytosol and are among the most potent cytotoxins known. Ribosome-inactivation is achieved in all cases through the cleavage of an N-glycosidic bond between ribose and a specific adenine residue in the universally conserved sequence 5xe2x80x2-AGUACGA*GAGGA-3xe2x80x2 (where A* indicates the target adenine) (SEQ ID NO: 164) located 250-400 nt from the 3xe2x80x2 end of 23S/25S/28S rRNAs (Endo and Tsurugi, 1987), (Hartley et al., 1996). Ribosomes depurinated in this manner are unable to bind the EF-2/GTP complex and protein synthesis is blocked at the translocation step (Montanaro et al., 1975). A single RIP molecule is able to depurinate 1000-2000 mammalian cell ribosomes per min under physiological conditions (Eiklid et al., 1980; Endo and Tsurugi, 1988).
proteins and enzymes such as RNase A that are potent cytotoxins (Leland et. al., 1998). These cytotoxic ribonucleases degrade cellular RNA and cause cell death and can be used to kill infected cells and so prevent the proliferation and spread of a pathogen.
These are examples of proteins which will inhibit the replication of a pathogen at a RNA, DNA or protein level by either binding directly to a pathogen protein, replication intermediate or a host factor that is necessary for pathogen replication or movement or transmission and the pathogen life cycle. This strategy is particularly suitable for inactivating viral pathogens. In addition, toxins, such as RIPs or RNase A are described that are suitable for causing cell death on pathogen entry and so halting the spread of infection or proliferation of a pathogen.
In principle all antibodies, proteins, peptides and enzymes that have an activity, that may or may not be enzymatic, which are able to interfere with pathogen life cycles are suitable as part of the present constructs.
In a preferred embodiment of the present invention said enzyme is chitinase or glucanase, glucose oxidase, superoxide dismutase, DNAse or RNAse or RIP or active fragments thereof either singly or in any combination(s).
Gene constructs may comprise the following or any combination of the follow and may be encoded on one or more plasmids: Gene constructs may comprise a nucleotide sequence or nucleotide sequences encoding complete recombinant full-size antibodies, dimeric secretory IgA antibodies, multimeric IgM antibodies, the F(abxe2x80x2)2 fragment, the Fab-fragment, the Fv-fragment, single chain antibody fragments (scFvs), single binding domains (dAbs), a bivalent scFv (diabody) (Poljak, 1994), minibody (Carter and Merchant, 1997), bispecific scFv antibodies (Plxc3xcckthun and Pack, 1997; Fischer et al. Eur. J. Biochem. 262, 810-816 (1999)) where the antibody molecule recognises two different epitopes that may come from the pathogen or the host or both, triabodies and any other part of the antibody (molecular recognition units (MRUs)) which shows binding to the target epitopes. Genes encoding these suitable recombinant antibody fragment(s) may be derived from vertebrates, camelids, avian or pisces species.
In the constructs according to the invention, the antibody is preferably fused to a complete sequence of a toxic agent or a part thereof which still has activity, or which is still functionally active. Also, the chimeric protein may be encoded by nucleotide sequences on one or more constructs and may be assembled in vivo by the plant or expression organisms protein assembly and translation machinery. The chimeric protein can also be obtained by biochemical assembly or in vitro or in vivo assembly of the chimeric immunotoxin subunits using the cells endogenous protein assembly machinery. The antibody, antibodies or fragments thereof are fused directly to the toxic agent or linked by a flexible spacer which does not interfere with the structure or function of the two proteins. Such flexible linkers include copies of the (Glycine-Glycine-Glycine-Glycine-Serine)n linker (SEQ ID NO: 165xe2x80x94also referred to as Gly4Ser), where n is 1 to 4 or more copies of the linker unit, the Genex 212 and 218 linker and the flexible linker peptide of Trichoderma reesi cellobiohydrolase I (CBHI) (Turner et al., 1997), (Tang et al., 1996).
In this invention, this targeting approach has the advantage that the molecular pathogenicide or antibody or fragment thereof can be expressed where the pathogen is most vulnerable to the action of the molecular pathogenicide and/or antibody or fragment thereof.
The desired cellular location of the molecular pathogenicide, or any components thereof, can be achieved by using the appropriate cellular targeting signals, these include but are not limited to signal peptides, targeting sequences, retention signals, membrane anchors, post translational modifications and/or membrane transmembrane domains that target the protein to the desired organelle, desired membrane (plasma membrane, ER, Golgi, nucleus, chloroplast or vacuole) or desired membrane orientation (cytoplasmic or lumenal or plant cell membrane display) (Kim et al., 1997; Rose and Fink, 1987). Localisation sequences can be targeting sequences which are described, for example in chapter 35 (protein targeting) of L. Stryer Biochemistry 4th edition, W.H. Freeman, 1995. Proteins synthesised without a functional signal peptide are not co-translationally inserted into the secretory pathway and remain in the cytosol. Proteins that carry a signal peptide that directs them to the secretory pathway, which may include a transmembrane sequence or membrane anchor, will be targeted for secretion by default or reside in their target membrane organelles. Targeting signals can direct proteins to the ER, retain them in the ER (LYSLYS motif (SEQ ID NO: 166) and KDEL (SEQ ID NO: 167)), TGN 38, or will target proteins to cell organelles such as the chloroplasts, vacuole, nucleus, nuclear membrane, peroxisomes and mitochondria. Examples for signal sequences and targeting peptides are described in (von Heijne, 1985) (Bennett and Osteryoung, 1991) (Florack et al., 1994). In addition, the targeting signals may be cryptic and encoded by a host plant cell or heterologous eukaryotic cell proteins or animal proteins where the localisation is known and where the protein can be cloned. By constructing a fusion protein with this protein, a molecular pathogenicide can be targeted to the localisation of the protein without the need for identification of the cryptic targeting signal. Suitable cryptic signals are those encoded by the resident Golgi enzymes.
The molecular pathogenicidds described in this invention can be targeted to cellular membranes by incorporating heterologous sequences into the recombinant protein which permit its synthesis as a membrane protein or as a membrane associated protein or its post translational modification to associate it with cellular membranes. Suitable membrane anchor Sequences, enabling the integration of recombinant antibody fusion proteins and tarts thereof in the plasma membrane, include the human T cell receptor transmembrane domains (Gross and Eshhar, 1992), glyco-phosphatidyl inositol (GPI) anchors (Gerber et al., 1992), immunoglobulin superfamily membrane anchors, tetraspan family members (Tedder and Engel, 1994; Wright and Tomlinson, 1994) and any transmembrane sequence(s) from a known protein or synthesised sequences that have a-similar function and can be included in the target protein by recombinant DNA technology.
In addition, the antibodies or parts thereof, or the recombinant antibody fusion proteins, or parts thereof, may be targeted to cell membranes where they could face the cytosolic side of the membrane. Suitable targeting sequences for cytoplasmic display, include the transmembrane domains of: KAR1, for nuclear membrane integration (Rose and Fink, 1987), middle-T antigen (Kim et al., 1997), for plasma membrane integration and cytochrome b5, for ER membrane integration (Kim et al., 1997). C-terminal linkages to fatty acids using consensus amino acid sequences leading to post translational prenylation, farnesylation, palmitoylation, myristoylation or ankyrin sequence motifs can also be used.
Pathogen-specific recombinant antibodies can be fused to different transmembrane anchors to improve the expression levels and stability of these molecules inside the plant cell, by targeting the expressed recombinant protein to cell membranes in various orientations. This can be accomplished by adding:
a) C-terminal localisation sequences to target and integrate recombinant cytosolic proteins with N-terminal leader peptides into the bilayer of cellular membranes, thus facing to the plant apoplast. Suitable membrane, localisation sequences include the human T cell receptor xcex2 chain transmembrane domain and the human platelet derived growth factor receptor (PDGFR) transmembrane domain, glyco-phosphatidyl inositol (GPI) anchors, immunoglobulin superfamily membrane anchors and any transmembrane sequence(s) from a known protein or synthesised sequences that have a similar function and can be included in the target protein by recombinant DNA technology.
b) Amino terminal transmembrane proteins with either dual or tetrameric plasma membrane spanning domains to expose both the N- and C-termini of secretory recombinant proteins to the cytosol. This can be achieved by using suitable members of the tetraspan family including CD9, CD20, CD81 and the In-Hc-lc dualspan typeII-IV hybrid of the MHC invariant chain and H-2d hybrid protein. This method enables the orientation of a secreted and membrane anchored antibody construct with its N- and C-terminus into the cytosol. Alternatively fusions to SNAP-25 can be used for the same orientation.
c) C-terminal anchor sequences to target and integrate recombinant cytosolic proteins without N-terminal leader peptides into the bilayer of endomembranes posttranslationally. Suitable targeting sequences include transmembrane domains of KAR1 for nuclear membrane integration (Rose and Fink, 1987), middle-T antigen for plasma membrane integration (Kim et al., 1997) and cytochrome b5 for ER membrane integration (Kim et al., 1997).
d) Addition of consensus motifs to the protein that permit C-terminal linkages to fatty acids by prenylation, farnesylation, palmitoylation, myristoylation in the cytosol which then lead to membrane integration.
e) Addition of ankyrin sequence motifs (Lambert and Bennett, 1993; Peters and Lux, 1993).
In addition, the antibody or fragment thereof can be encoded by a separate nucleotide sequence to that of the toxin and the antibody and toxin, either of which may encode membrane localisation or cellular targeting sequences, can be encoded by one or more vectors, e.g., plasmids. The constructs contain nucleotide sequences encoding complimentary binding proteins so that when the antibody, or fragment thereof, is genetically fused to one binding partner and the toxin, or fragment thereof, is genetically fused to the second binding partner, these two independent proteins will bear mutually recognising binding activities. When these two independent proteins are expressed in the same plant compartment, the binding domains will bind to form a molecular pathogenicide with two subunits and similar properties to an antibody-toxin fusion protein. Suitable binding domains/partners include:
A single chain antibody and its corresponding epitope, where the single chain binds to the epitope and thereby enables binding between two independent proteins,
leucine zippers (Carter et al., 1995),
Antibody heavy and light chains, where one protein is fused to the heavy chain and assembly of heavy and light chain takes place in the ER,
other homo- or hetero-binding domains.
Anyone of ordinary skill in the art will recognise that the component antibody, antibodies or fragments thereof or component pathogen binding peptides, as described, and component toxin or fragments thereof can each bear a binding partner. When expressed in the same compartment of a plant, or when encountering each other, these binding domains can then permit the assembly of a molecular pathogenicide with all the properties required from the components. Anyone skilled in the art will recognise that this can be achieved by other means than those described above which are intended as examples to better illustrate the principle of in vivo assembly and are not intended to be taken as a limiting or a comprehensive description.
Anyone skilled in the art will also recognise that the various components of the present invention can be expressed in such a way that they are on the surface of a third carrier protein, suitable carriers include glutathione S-transferase (GST) encoded by Schistosoma japonicum (Smith and Johnson, 1988), TMV coat protein, maltose-binding protein and thioredoxin (LaVallie et al., 1993) or other proteins. In addition, any of the components of the present invention may be tagged with a genetically encoded fluorophore, suitable fluorophores include, but are not limited to, the green fluorescent protein (GFP) from Aequonria victoria. This approach would be especially useful for monitoring the localisation of a pathogen or molecular pathogenicide during infection.
If the fusion protein or proteins are expressed in a heterologous organism for production of the protein or proteins, it may be necessary to modify the gene construct in order to match the codon preference of the organism and to remove mRNA motifs that reduce the stability of the transcript.
All of the components of the molecular pathogenicides described in this invention can be separately transformed into plant lines which can then be sexually crossed to give offspring that product the molecular pathogenicides in a functional form.
Anyone skilled in the art will recognise that the antibodies, peptides and toxins can be combined in several forms and encoded on different plasmids to produce proteins that have the desired effect on the pathogen. Anyone skilled in the art will also recognise that assembling the molecular pathogenicides from individually genetically encoded subunits can be achieved by several methods.
Viruses, bacteria, mycoplasmas, fungi, nematodes, insects and other pathogens.
The present invention also relates to vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain a polynucleotide according to the invention or any one of the above-described gene constructs. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
In a preferred embodiment, the polynucleotide present in the vector is linked to regulatory elements which allow the expression of the polynucleotide in prokaryotic and/or eukaryotic cells. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic and/or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilisation of the transcript, for example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV). In this respect, the person skilled in the art will readily appreciate that the polynucleotides encoding at least one of the above-described domains of the fusion proteins or pathogenicide of the invention may encode all of the domains or only one. Likewise, said polynucleotides may be under the control of the same promoter or may be separately controlled for expression. Other promoters commonly used are the Figwort Mosaic virus promoter, the polyubiquitin promoter, and the actin promoter for ubiquitous expression. The termination signals usually employed are from the Nopaline Synthase or CaMV 35S gene. A plant translational enhancer often used is the TMV omega sequences, the inclusion of an intron (Intron-1 from the Shrunken gene of maize, for example) has been shown to increase expression levels by up to 100-fold. (Maiti et al., Transgenic Research 6 (1997), 143-156; Ni et al., Plant Journal 7 (1995), 661-676). Additional regulatory elements may include transcriptional as well as translational enhancers.
Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL). Advantageously, the above-described vectors of the invention comprises a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed hosts, for example plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable genes have been described, namely trpB, which allows cells to utilise indole in place of tryptophan; hisD, which allows cells to utilise histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilise mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, said marker is a gene encoding luciferase (Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or xcex2-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a vector of the invention.
The present invention furthermore relates to host cells comprising a vector as described above or a polynucleotide according to the invention. The vector or polynucleotide according to the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extrachromosomally.
The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. 
Another subject of the invention is a method for the preparation of the above-described fusion proteins and pathogenicides which comprises the cultivation of host cells according to the invention which, due to the presence of a vector or a polynucleotide according to the invention, are able to express such a protein, under conditions which allow expression and optionally assembly of the fusion protein or pathogenicide and recovering of the so-produced protein from the culture. Depending on the specific constructs and conditions used, the protein may be recovered from the cells, from the culture medium or from both. For the person skilled in the art it is well known that it is not only possible to express a native protein but also to express the protein as fusion polypeptides or to add signal sequences directing the protein to specific compartments of the host cell, e.g., ensuring secretion of the peptide into the culture medium, etc. Furthermore, such a protein and fragments thereof can be chemically synthesised and/or modified according to standard methods described, for example herein.
The present invention furthermore relates to molecular pathogenicides encoded by the polynucleotides according to the invention or produced by the above-described method. In this context, it is also understood that the fusion proteins and pathogenicides according to the invention may be further modified by conventional methods known in the art.
The fusion constructs are expressed in plants either stably in transgenic plants or transiently under the control of any type of promoter that is active in plants. For long-term resistance in host plants, high yield production of recombinant proteins, stable expression is preferred.
In general, such regulatory elements comprise a promoter active in plant cells. To obtain expression in all tissues of a transgenic plant, preferably constitutive promoters are used, such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689). In order to achieve expression in specific tissues of a transgenic plant it is possible to use tissue specific promoters (see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251). Further examples are:
a) Expression control elements (e.g. promoters listed below in b to f, enhancer sequences, transcriptional and translational enhancers, transcription terminators, polyadenylation sites etc.) and a selectable marker if necessary.
b) Constitutive promoters such as the CaMV-35S (Benfey et al., 1989) and the nos promoter (Mitra and Gynheung, 1989).
c) Viral subgenomic promoters.
d) Tissue specific promoters and chimeric promoters (Ni et al., 1995), (Comai et al., 1990).
e) Inducible promoters (Caddick et al., 1998).
f) Transient expression systems (Kapila et al., 1997).
Known are also promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley etc. Inducible promoters may be used in order to be able to exactly control expression. An example for inducible promoters are the promoters of genes encoding heat shock proteins. Also microspore-specific regulatory elements and their uses have been described (WO96/16182). Furthermore, the chemically inducible Tet-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991); 229-237). Further suitable promoters are known to the person skilled in the art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993), 361-366). The regulatory elements may further comprise transcriptional and/or translational enhancers functional in plants cells. Furthermore, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability.
Furthermore, it is in principle possible to modify the coding sequence in such a way that the protein is located in any desired compartment of the plant cell. These include the endoplasmatic reticulum, the vacuole, the mitochondria, the plastids, the apoplast, the cytoplasm etc. Methods how to carry out this modifications and signal sequences ensuring localisation in a desired compartment are well known to the person skilled in the art.
Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment and other methods known in the art. The vectors used in the method of the invention may contain further functional elements, for example xe2x80x9cleft borderxe2x80x9d- and xe2x80x9cright borderxe2x80x9d-sequences of the T-DNA of Agrobacterium which allow for stably integration into the plant genome. Furthermore, methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e. the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example cotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161; Peng, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems which utilise enzymes capable of promoting homologous recombination in plants (see, e.g., WO097/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361); Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378). Methods for the preparation of appropriate vectors are described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976; Koncz, Specialised vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example if a phenotype conferred by said strain is desired.
Methods for the transformation using biolistic methods are well known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1, 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, N.Y. (1995).
The transformation of most dicotyledonous plants is possible with the methods described above. But also for the transformation of monocotyledonous plants several successful transformation techniques have been developed. These include the transformation using biolistic methods as, e.g., described above as well as protoplast transformation, electroporation of partially permeabilized cells, introduction of DNA using glass fibers, etc.
Transformation can be done using any method that leads to expression of construct or constructs in a plant and these methods can be used for stable transformation where the gene of interest is incorporated in the host plant DNA or where the construct is transiently expressed. Examples of transformation technology include:
a) Agrobactetum tumefaciens or Agrobacterium rhizogenes mediated transformation (Turpen et al., 1993; White, 1992): based on the insertion of a foreign DNA sequence into the plant genome carried on a plasmid DNA within the agrobacteria. The foreign gene is inserted into the plant genome together with bacterial plasmid sequences.
b) Particle bombardment (Sanford et al., 1990), (Klein and Fitzpatrick-McElligott, 1993) or biolistic process (Furth, 1997): Particle bombardment uses particles coated with the DNA that penetrate the plant cell at high velocity and the DNA is incorporated into the host genome by host recombination processes. Besides particle bombardment biolistic processes also include injection methods.
c) Tissue electroporation (Chowrira et al., 1995; D""Halluin et al., 1992): under the influence of an electric field, DNA enters pores in the plant cell membrane and is incorporated into the plant genome by recombination.
d) Use of liposomes or methods which increase the uptake of free DNA (Spxc3x6rlein and Koop, 1991; White, 1992).
e) Any method for integration of foreign DNA in a plant cell resulting in transiently or stably transformed plants.
The present invention relates to transgenic plant cells which contain a polynucleotide, vector or composition of vectors of the invention. Preferably, said polynucleotide or vector is stably integrated into the genome.
As is immediately evident to the person skilled in the art, the vectors of the present invention can carry nucleic acid molecules encoding the domains of the antibody, fusion protein or pathogenicide of the invention either alone or in combination. The same applies to the above described plant cells, plant tissue and plants transformed therewith. Likewise, said nucleic acid molecules may be under the control of the same regulatory elements or may be separately controlled for expression. In this respect, the person skilled in the art will readily appreciate that the nucleic acid molecules encoding the domains of the fusion protein or pathogenicide can be expressed in the form of a single mRNA as transcriptional and optionally translational fusions. This means that domains are produced as separate polypeptides or in the latter option as a fusion polypeptide that is further processed into the individual proteins, for example via a cleavage site for proteinases that has been incorporated between the amino acid sequences of both proteins. The resultant protein domains can then self-assemble in vivo. Of course, the domains may also be expressed as a bi- or multifunctional polypeptide, preferably disposed by a peptide linker which advantageously allows for sufficient flexibility of both proteins. Preferably said peptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of one of said proteins and the N-terminal end of the other of said proteins when said polypeptide assumes a conformation suitable for biological activity of both proteins when disposed in aqueous solution in the plant cell. Examples of the above-described expression strategies can be found in the literature, e.g., for dicistronic mRNA (Reinitiation) in Hefferon, J. Gen. Virol, 78 (1997), 3051-3059, fusion proteins are described in Brinck-Peterson, Plant Mol. Biol. 32 (1996), 611-620 and Hotze, FEBS Lett. 374 (1995), 345-350; bifunctional proteins are discussed in Lamp, Biochem. Biophys. Res. Corn. 244 (1998), 110-114 and Dumas, FEBS Lett. 408 (1997), 156-160 and for linker peptide and protease it is referred to Doskeland, Biochem. J. 313 (1996), 409-414.
In a preferred embodiment of the invention, the transgenic plant cell comprises a selectable marker. As described above, various selectable markers can be employed in accordance with the present invention. Advantageously, selectable markers may be used that are suitable for direct selection of transformed plants, for example, the phophinothricin-N-acetyltransferase gene the gene product of which detoxifies the herbicide L-phosphinothricin (glufosinate or BASTA); see, e.g., De Block, EMBO J. 6 (1987), 2513-2518 and Drxc3x6ge, Planta 187 (1992), 142-151.
The presence and expression of the polynucleotides or vectors in the transgenic plant cells leads to the synthesis of a fusion protein, antibody or pathogenicide of the invention or assembly of the same which has an influence on pathogen resistance in plants containing such cells.
Thus, the present invention also relates to transgenic plants and plant tissue comprising transgenic plant cells according to the invention. Due to the expression of a fusion protein, the antibody against the viral movement and/or replicase protein or pathogenicide of the invention or their domains, e.g., in cellular compartments and/or plant tissue these transgenic plants may show various physiological, developmental and/or morphological modifications in comparison to wild-type plants. Advantageously, these transgenic plants display a resistance against a pathogen that the corresponding wild type plant was susceptible to.
In general, the plants which can be modified according to the invention can be derived from any desired plant species. They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture, such as crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oil producing plants (e.g. oilseed rape, sunflower, pea nut, soy bean, etc.), cotton, sugar beet, sugar cane, leguminous plants (e.g. beans, peas etc.), wood producing plants, preferably trees, etc.
In yet another aspect, the invention also relates to harvestable parts and to propagation material of the transgenic plants according to the invention. Harvestable parts can be in principle any useful parts of a plant, for example, leaves, stems, fruit, flowers, seeds, roots etc. Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
In addition, the present invention relates to a kit comprising the above-described antibodies, fusion protein, pathogenicide, polynucleotide or vectors. The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic plant cells, plant tissue or plants. The kit of the invention may advantageously be used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool. The parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art. The kit or its ingredients according to the invention can be used in plant cell and plant tissue culture, for example in agriculture. The kit of the invention and its ingredients are expected to be very useful in breeding new varieties of, for example, plants which display improved properties such as those described herein.
It is also immediately evident to the person skilled in the art that the polynucleotides and vectors of the present invention can be employed to produce transgenic plants with a further desired trait due to genetic engineering (see for review TIPTEC Plant Product and Crop Biotechnology 13 (1995), 312-397). This can be, for example, an acquired resistance to other pathogens or quality improvements of the plants comprising (i) herbicide tolerance (DE-A-3701623; Stalker, Science 242 (1988), 419), (ii) insect resistance (Vaek, Plant Cell 5 (1987), 159-169), (iii) virus resistance (Powell, Science 232 (1986), 738-743; Pappu, World Journal of Microbiology and Biotechnology 11 (1995), 426-437; Lawson, Phytopathology 86 (1996), 56 suppl.), (vi) ozone resistance (Van Camp, BioTech. 12 (1994), 165-168), (v) improving the preserving of fruits (Oeller, Science 254 (1991), 437-439), (vi) improvement of starch composition and/or production (Stark, Science 242 (1992), 419; Visser, Mol. Gen. Genet. 225 (1991), 289-296), (vii) altering lipid composition (Voelker, Science 257 (1992), 72-74), (viii) production of (bio)polymers (Poirer, Science 256 (1992), 520-523), (ix) alteration of the flower colour, e.g. by manipulating the anthocyanin and flavonoid biosynthetic pathway (Meyer, Nature 330 (1987), 667-678, WO90/12084), (x) resistance to bacteria, insects and fungi (Duering, Molecular Breeding 2 (1996), 297-305; Strittmatter, Bio/Technology 13 (1995), 1085-1089; Estruch, Nature Biotechnology 15 (1997), 137-141), (xi) inducing and maintaining male and/or female sterility (EP-A1 0 412 006; EP-A1 0 223 399; WO93/25695) and (xii) remediation of contaminated soils (Cunningham, TIBTECH 13 (1995), 393-397).
These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public data base xe2x80x9cMedlinexe2x80x9d may be utilised which is available on the Internet, for example at the following World Wide Web domain site address xe2x80x9cncbl.nlm.nih.gov/PubMed/medline.htmlxe2x80x9d. Further databases and addresses such as those found at the World Wide Web domain site addresses xe2x80x9cncbi.nlm.nih.govxe2x80x9d, xe2x80x9cinfoblogen.frxe2x80x9d, xe2x80x9cfmi.ch/biology/research_tools.htmlxe2x80x9d, and xe2x80x9ctigr.orgxe2x80x9d are known to the person skilled in the art and can also be obtained using search engines such as xe2x80x9clycos.comxe2x80x9d. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness if given in Berks, TIBTECH 12 (1994) 352-364.